The long term objective of this proposed research is to understand the function of mammalian transcription factors that interact with RNA Polymerase II. Such factors have been isolated by affinity chromatography on calf thymus RNA polymerase II immobilized on an agarose column. The particular focus of this grant is to develop reagents and assays to determine the function of RAP74 (RNA polymerase II-associating protein; 74 kilodaltons) in transcriptional initiation. RAP 74 interacts with another RAP of 30 kilodaltons that is known to be a general initiation factor. The RAP30/74 complex is associated with an ATP-dependent DNA helicase, which is speculated to separate template strands, allowing formation of phosphodiester bonds. Unlike many other RNA polymerases, RNA polymerase II requires ATP hydrolysis in order to initiate a chain. Preliminary data indicate that RAP74 is the ATP hydrolyzing function in initiation. The essential advance in studying the RAPs was the cloning of a cDNA encoding human RAP30. A cDNA encoding human RAP74 is now being isolated. RAP genes are used to generate the necessary sequence data and reagents to determine RAP function. Transcription is regulated in mammalian systems by sequence-specific DNA binding factors that stimulate a rate-limiting step in the general initiation mechanism, in which RAP30/74 participates. Both RAP30 and RAP74 are phosphoproteins, so phosphorylation could influence their interaction with upstream sequence or Z enhancer-binding factors, with each other and with polymerase. Thus, regulation of gene expression may involve modification of RAP30/74. Some oncogenes are enhancer-binding factors and some participate in phosphorylation cascades that may modify transcription factors. Therefore, regulated contacts between RAP30/74 and other transcription factors may be an important molecular mechanisms in cell transformation.